Study On The Effect And The Mechanism Of MiR-126 On Cerebral Angiogenesis

Objective: To investigate the effect of EGFL7 mediated by microRNA126 on angiogenesis in human neural stem cells(NSCs)and its mechanism.Methods:In this study,human umbilical vein endothelial cells(HUVEC)and nerve stem cells(NSCs)were used as research objects to construct recombinant vectors of microRNA-126 mimic and inhibitor,verify and transfect cells,confirm the transfection rate under fluorescence microscope,and then carry out the following experiments: 1.Double luciferase reporter gene experiment to verify the relationship between microRNA-126 and EGFL7;2.CCK-8 method to detect transfected carriers.3.Detection of the expression of intracellular microRNA126 and EGFL7 after transfection by real-time fluorescence quantitative polymerase chain reaction(q RT-PCR)and Western Blot;4.Transwell chamber technique was used to co-culture NSCs and HUVEC,and the effect of transfection vector on the migration ability of HUVEC cells was observed under the co-culture condition by microscope,so as to further clarify the effect of transfection vector on the tubulogenic ability of HUVEC cells.5.Western Blot technique was used to detect the expression of PI3K/AKT pathway-related proteins in HUVEC before and after vector transfection.Results: 1.The recombinant vectors of microRNA-126 mimic and microRNA-126 inhibitor were successfully constructed and transfected into HUVEC cells.The transfection rate of the recombinant vectors in HUVEC cells was about 70% by fluorescence microscopy.The double luciferase reporter gene assay showed that EGFL7 was the target gene of microRNA-126.2.The results of cell proliferation test showed that the proliferation of HUVEC cells transfected with miR-126 mimic was inhibited compared with that of control group(P < 0.05),while the proliferation of HUVEC cells transfected with miR-126 inhibitor was higher than that of control group(P < 0.05).3.The results of q RT-PCR showed that the expression of EGFL7 gene in HUVEC cells of miR-126 mimic group was significantly lower than that of control group(P < 0.05),and the expression of miR-126 was higher than that of other groups(P < 0.05),while the expression of EGFL7 gene in HUVEC cells of miR-126 inhibitor group was significantly higher than that of control group(P <0.05),and the expression of miR-126 was lower than that of other groups(P < 0.05).The results showed that the expression of EGFL7 in the microRNA-126 mimic group was lower than that in the control group,while the expression of EGFL7 in the microRNA-126 inhibitor group was not inhibited.4.Under co-culture conditions,HUVEC cell migration level and tubuloinduction ability in the microRNA-126 mimic group were lower than those in the control group.The level of cell migration in the microRNA-126 inhibitor group was higher than that in the control group,and the ability of cell tube formation was significantly enhanced.5.Western Blot results showed that the expression of PI3 K,P-PI3 K,AKT and P-AKT proteins in the microRNA-126 inhibitor group were all decreased(P < 0.05),while the expression of these pathway proteins in the microRNA-126 inhibitor group was significantly increased(P < 0.05).Conclusions:This study suggests that EGFL7 is involved in microangiogenesis in the co-culture system of neural stem cells and vascular endothelial cells and is regulated by microRNA-126,which may be mediated by EGFL7/PI3K/AKT signaling pathway.